Cancer And Approaches To Its Therapy
Cancer results when normal cells undergo neoplastic transformation and develop into malignant tumors. This transformation is due to underlying genetic alterations. Oncogenes are believed to be altered forms of normal genes, called proto-oncogenes, that act as central regulators of growth in normal cells. When a carcinogenic agent such as radiation or a chemical carcinogen, damages the DNA of the target gene of a cell, cancer may develop. Once activated by a mutation, an oncogene may promote excessive or unregulated cell growth. One class of oncogenes acts in normal cells to suppress rather than to promote cell proliferation. A loss of this type of growth suppressor gene from a cell removes a normal constraint on cell growth, leading to uncontrolled proliferation, which in turn may lead to cancer.
Intensive efforts to develop therapies which can prevent or block the development of cancer are currently under way. Historically, efforts have been focused on the treatment of disease using surgery, radiotherapy, and various forms of chemotherapy, to remove or destroy the tumor tissues. The cytotoxic methods are severely limited by their lack of specificity.
Immunotoxins and Their Limitations
Immunotoxins have been developed by conjugating a protein toxin to a tumor-specific monoclonal antibody via a linker for targeted tumor therapy (Vitetta, E. S. et al., Ann. Rev. Immunol. 3:197-212 (1985)). In principle, an injected immunotoxin is transported through the blood stream to the targeted tumor tissue, penetrates the tissue, binds to the individual tumor cells, and the toxin acts in a highly localized manner to destroy only the bound tumor cells. All three components of the conjugates are important for the specific delivery of the cytotoxicity: The antibody enables the conjugate to be retained in the target tissue by binding to a specific cell-surface antigen, which enhances cellular uptake by the target cells. The linker keeps the toxin bound to the antibody and inactive while in circulation, but allows for rapid release of the active toxin inside the target cells. The toxin kills the cell by inhibiting cellular protein synthesis, or by some other related mechanism.
Some of the most cytotoxic substances known are protein toxins of bacterial and plant origin (Frankel, A. E. et al., Ann. Rev. Med. 37:125-142 (1986)). The cytotoxic action of these molecules involves two events--binding the cell surface and inhibition of cellular protein synthesis. The most commonly used plant toxins are ricin and abrin; the most commonly used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin A.
In ricin and abrin, the binding and toxic functions are contained in two separate protein subunits, the A and B chains. The ricin B chain binds to the cell surface carbohydrates and promotes the uptake of the A chain into the cell. Once inside the cell, the ricin A chain inhibits protein synthesis by inactivating the 60S subunit of the eukaryotic ribosome Endo, Y. et al., J. Biol. Chem. 262:5908-5912 (1987)).
Diphtheria toxin and Pseudomonas exotoxin A are single chain proteins, and their binding and toxicity functions reside in different domains of the same protein chain. In diphtheria toxin, the C-terminal domain inhibits protein synthesis by ADP-ribosylation of the elongation factor, EF2. The two activities are separate, and the toxin elicits its full activity only after proteolytic cleavage between the two domains. Pseudomonas exotoxin A has the same catalytic activity as diphtheria toxin.
The use of diphtheria toxin-based immunotoxins is limited by the fact that most people have been immunized against diphtheria toxin. The use of ricin-based immunotoxins is also limited because these immunotoxins exhibit specific toxicity only in the presence of lactose, which at high concentrations competes with the cell surface carbohydrates for the B chain binding sites. An alternative approach has been developed to use ricin A chain or "single chain ribosome inactivating protein" (SCRIP) in the preparation of immunotoxins.
Single Chain Ribosome Inactivating Proteins (SCRIPs) and Their Potential Application in Tumor Therapy
SCRIPs are highly active at inactivating ribosomes in cell-free systems, but are relatively nontoxic to intact cells. A wide variety of such molecules are found in plants. These include pokeweed antiviral protein, wheat germ protein, gelonin, dianthins, momorcharins, trichosanthin, and many others (Strip, F. et al., FEBS Lett. 195:1-8 (1986)). Some of these SCRIPs have been exploited in the preparation of immunotoxins. Once inside the cell, their cytotoxicity is surprisingly higher than that of the native "holo" counterparts. Many of these SCRIPs are antiviral agents, and some also exhibit specific antitumor activity.
HIV Infection and AIDS
Human Immunodeficiency Virus (HIV), the etiological agent for AIDS (Acquired Immune Deficiency Syndrome), is a member of the lentiviruses, a subfamily of retroviruses. Many retroviruses are well-known carcinogens. HIV per se is not known to cause cancer in humans or other animals, but it does present a formidable challenge to the host. HIV integrates its genetic information into the genome of the host. The viral genome contains many regulatory elements which allow the virus to control its rate of replication in both resting and dividing cells. Most importantly, HIV infects and invades cells of the immune system; it destroys the body's immune system and renders the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
HIV is transmitted by parenteral inoculation and/or intimate sexual contact. It is estimated that about 2 million people in the United States are currently infected with HIV, and 5 to 10 million people are infected worldwide. Recent projections indicate that a majority of those now infected will develop AIDS within a seven year follow-up period. In 1989 alone, over 130,000 cases of AIDS were reported domestically, and more than half of these patients have died. It is estimated that an additional 200,000 cases will be diagnosed in the United States by the end of 1990. Reports to the World Health Organization suggest that at least a million of new cases of AIDS can be expected within the next five years worldwide. It is apparent that AIDS is an unprecedented threat to U.S. as well as global health. The search for effective therapies to treat AIDS is of paramount importance.
HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system which express the cell surface differentiation antigen CD4 (also known as OKT4, T4 and leu3). The viral tropism is due to the interactions between the viral envelope glycoprotein, gp120, and the cell-surface CD4 molecules (Dalgleish, A. G. et al., Nature 312: 763-767 (1984). These interactions not only mediate the infection of susceptible cells by HIV but are also responsible for the virus-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death, and progressive depletion of CD4 cells in AIDS patients. These events result in HIV-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms.
In addition to CD4+ T cells, the host range of HIV includes cells of the mononuclear phagocytic lineage (Dalgleish, A. G. et al., supra), including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes. HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. Macrophage/monocytes are a major reservoir of HIV. They may interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
Anti-HIV Drugs
Intensive efforts are currently under way to develop therapies to prevent or intervene in the development of clinical symptoms in HIV-infected individuals. For the most part, efforts have been focused on the use of nucleoside analogue drugs such as AZT (azidothymidine), and on other dideoxynucleoside derivatives such as ddA (dideoxyadenosine), ddT (dideoxythimedine), ddI (dideoxyinosine), and ddC (dideoxycytidine). These drugs inhibit the viral enzyme, reverse transcriptase, thereby inhibiting de novo infection of cells. However, once viral infection has been established within a cell, viral replication utilizes host cell enzymes. Thus, drugs which inhibit only reverse transcriptase would be expected to have limited effects. While the spread of free virus within the organism may be blocked, the mechanisms of syncytium formation and pathogenesis through direct intercellular spread remain.
A very small number of HIV-infected T cells can fuse with, and eventually kill, large numbers of uninfected T cells through mechanisms based on viral surface antigen expression. In vitro studies have demonstrated HIV replication even in the continued presence of nucleoside analogues in prolonged culture. Drugs targeting other viral processes are also being developed, such as soluble CD4 and dextran sulfate to inhibit viral binding, alpha interferons and "ampligen" to inhibit viral budding, and castanospermine to inhibit the processing of the viral glycoproteins. These drugs are still in early stages of testing. The actual processes of HIV intracellular replication and protein synthesis have not been specifically targeted because these viral functions were thought to reflect the mere pirating of normal host processes through host mechanisms.
Recently, M. S. McGrath et al. (Proc. Natl. Acad. Sci. USA 86:2844-2848(1989)) reported that GLQ 223, a SCRIP isolated from the Chinese medicinal plant, Trichosanthis kirilowii, selectively inhibits HIV replication. This compound demonstrated dose-dependent anti-HIV activity in both acutely and chronically infected T cells, as well as in monocytes/macrophages. This discovery has led to the rapid clinical testing of GLQ 223 as an anti-AIDS drug. Treatment of cells with GLQ 223 resulted in selective inhibition of the synthesis of viral DNA, RNA, and protein, with little or no effect on cellular synthesis. Inhibition of viral replication occurred at GLQ 223 concentrations that had no detectable effect on uninfected cells.
The mechanisms of the selective anti-HIV activity of GLQ 223 is not known. It has not been established whether this activity is associated with the ribosome-inactivating or the abortifacient activity of this compound. Two possible mechanisms are immediately apparent. Selective binding or uptake of GLQ 223 by infected cells could be responsible for its selective action on infected cells. Once inside the infected cells, the compound could act non-specifically, via its ribosome inactivating function. Alternatively, selectivity of the agent may arise from differential effects on viral versus cellular components, resulting in inhibition of viral but not of cellular nucleic acid and protein synthesis.
Lifson et al., U.S. Pat. No. 4,795,739 (issued Jan. 3, 1989), discloses that plant proteins, including trichosanthin and alpha and beta Momorcharin (also known as Momorcharin A and B, or MCA and MCB), reduce viral antigen expression in HIV-infected cells, and are selectively toxic to HIV-infected cells. These proteins are said to be useful for treating HIV infections in humans.